Nerve growth factor (NGF) is required for the normal development of distinct subclasses of peripheral neurons and plays yet undefined roles in the development of the central nervous system. The cellular mechanisms subserving NGF's action on its target cells are largely unknown. In PC12 cells NGF rapidly activates the transcription of a group of "immediate early genes", including the protooncogenes c-fos and c-myc. These "immediate early genes" encode putative transcriptional regulators and are likely to be critically involved in the regulation of expression of other genes which are required for the acquisition of a neuronal phenotype. This proposal is directed at the identification of NGF regulated "immediate early genes" and establishing their role in the differentiation PC12 cells. We have constructed a cDNA library from PC12 role in the differentiation PC12 cells. We have constructed a cDNA library from PC12 cells treated for 30 min with NGF. The library was screened with a subtracted probe and approximately 30 NGF-regulated to GNF, and represent the first examples of hormone-specific "immediate early genes". Partial sequence analysis revealed no homologies to other known genes. We propose to further characterize and sequence these cDNA clones and investigate their response to NGF and other agents. We propose a strategy to investigate the biological function of the "immediate early genes" through creation of hypomorphic or null mutants in which specific gene products are deleted from the cells by inhibition of synthesis of the gene products by anti-sense oligodeoxynucleotides complementary to the mRNA of these genes. Oligonucleotides are rapidly taken up into the cells and provide a facile method of specifically blocking gene expression. Initially, we will investigate the role of the c-fos in the transcriptional regulation of genes which are normally induced at later times following treatment of the cells with NGF, as well as the effect of antisense oligonucleotides on cellular proliferation and morphological differentiation of the PC12 cells. In addition, the ability of c-fos to regulate its own transcription and that of other "immediate early genes" will be investigated. Once sequence information is available for the cDNA clones derived from the PC12 library the function of these gene products will be studied using antisense oligonucleotide-mediated deletion of the gene products from the cells.